Remarkable progress has occurred during the past decade in our understanding of the processes responsible for the production of thyroid hormones (TH), and the mechanisms whereby they exert their profound effects on cellular processes. During this period, our research efforts have focused on developing a better understanding of the biochemical processes responsible for TH metabolism. This is of considerable physiologic importance given that the principal product of the thyroid gland, T4, functions primarily as a prohormone and undergoes extensive metabolism in peripheral tissues to form both active and inactive metabolites. The process of deiodination, whereby an iodine is removed from the phenolic or tyrosyl ring of an iodothyronine compound, represents the principal mechanism of TH metabolism, and is catalyzed by a family of three enzymes termed the types 1, 2, and 3 deiodinases (D1, D2, D3, respectively). In the past ten years, our laboratory, in collaboration with others at our own and other institutions, has been instrumental in isolating and characterizing the first cDNAs for each of the three deiodinase isoforms. The information and reagents derived from these studies now provide us with the opportunity to gain greater insight into the workings of these enzymes in their native intracellular environment. Thus, the specific aims of this application are: (1) To analyze the functional characteristics of the D1, D2, and D3 in situ. Studies will be undertaken to characterize the catalytic properties of the deiodinases in intact cultured cells and those permeabilized to allow ready entry of substrates, cofactors and other reagents. (2) To determine the intracellular location and topographic orientation of the D1, D2, and D3 in situ. This will be accomplished in cultured cells using immunocytochemical techniques and chimeric proteins whereby the D1, D2, and D3 are tagged with green fluorescent protein or other epitopes. (3) To define the post-translational mechanism(s) regulating D1 and D2 acti vity. Studies utilizing specific antiserum and epitope- tagged constructs will determine the intracellular trafficking and degradation rates of the D1 and D2 and how these are affected by the presence of TH and other reagents. (4) To determine the relative efficiency of the D1 versus the D2 in regulating nuclear T3 levels. Using a transgenic mouse model whereby D2 is targeted for expression in the liver, we will undertake studies to examine the degree to which the D1 and the D2 contribute to the nuclear pool of T3. These studies should provide important new insights into the biochemistry and physiologic relevance of the different deiodinase isoforms.